Both cell cycle checkpoints and DNA damage repair are essential elements for maintaining genome integrity and preventing tumorigenesis. However, the mechanisms whereby checkpoints are activated and coordinated with DNA damage repair at the molecular level are not well defined. Nbs1, which is linked to the Nijmegen Breakage Syndrome (NBS), plays an essential role in maintaining genome stability. Loss of Nbs1 function leads to radiation sensitivity, chromosomal instability and cancer. Nbs1 forms a tight complex with two recombination proteins Mre11 and Rad50, and functions in both S-phase checkpoint control and DNA repair. Additionally, our recent studies suggest a potentially novel role for Nbs1 in preventing multiple rounds of DNA replication in a single cell cycle. We propose to investigate how DNA damage signals are transduced to Nbs1 to subsequently activate different cellular responses through Nbs1 in a coordinated manner. First, we will characterize Nbs1 phosphorylation events triggered by various genotoxic stresses and define which kinases are important for Nbs1 phosphorylation. Second, we will investigate whether phosphorylation and other cellular interactions are important for Nbs1 function in DNA damage repair. Third, we will elucidate what role Nbs1 plays in mediating replication initiation and S-phase checkpoint control through shared biological pathways and we will determine how these two processes are regulated. These studies will establish detailed molecular pathways whereby the various functions of Nbs1 are activated and will allow us to gain significant insights into the overall mechanisms of how the checkpoint network functions to maintain genome stability. Our studies will ultimately help to develop therapeutic interventions for human diseases associated with genome instability.